Humangranulocyticanaplasmosis(HGA)isapotentiallyfataltick-borneinfection.Thesteepriseinthe numberofreportedcasesinrecentyears,evidencethatthetruenumberisgrosslyunderreported,and its potential for severe outcome make HGA an emerging disease and serious health concern. The etiologic agent is Anaplasma phagocytophilum (Ap), an obligate intracellular bacterium that invades neutrophils and endothelial cells. Like all obligate intracellular bacteria, Ap must enter host cells to survive.Identifyingthebacterialadhesinsandhostcellreceptorsthatmediatethisessentialprocessis fundamental to understanding Ap pathogenesis and for preventing infection. We have made great progressonthisfrontduringthepreviousfundingperiod.WeidentifiedthreeApadhesinscalledOmpA, Asp14,andAipAthataresufficientandnecessaryforoptimalinvasionofmyeloidandendothelialcells. Apupregulateseachoftheseattwocriticalstagesoftheinfectioncyclethatmakethemidealtargets forneutralizingantibodies:whenitconvertstotheinfectiousformthatinvadeshostcellsandduringthe tickbloodmealthattransmitsApintomammals.ThethreeadhesinsarehighlyconservedamongAp strains.Wedelineatedtheadhesins?essentialbindingdomainsasOmpAresidues59to74(OmpA59- 74),Asp14residues113to124(Asp14113-124),andAipAresidues9to21(AipA9-21).Anantibodycocktail targeting only these three binding domains blocks Ap infection of host cells in vitro. In terms of comprehensively dissecting each adhesin?s role in invasion, we achieved this first for OmpA by identifyingitsreceptoranddelineatingthetwoOmpAaminoacidsthatmediatetheinteraction.Weare now focused on Asp14 and AipA. We identified the Asp14 receptor as a host cell surface localized enzymeandconfirmedthatthisinteractionrequiresAsp14113-124.WhiletheAsp14receptorcontributes toApadhesion,itscellsurfaceenzymaticactivityisimportantforinvasion.Themechanismbywhichit doessoisundefined.WealsoidentifiedtwopromisingAipAreceptorcandidates.Forourcompetitive renewal,wewillbuildontheseexcitingdata.InAim1,wewilldissecttheAsp14-receptorinteraction anditsroleinApinfectioninvitroandinvivo.InAim2,wewillpinpointtheAipAreceptoranddefineits relevance to infection. In Aim 3, we will establish the importance of OmpA, Asp14, and AipA for Ap infectivityinvivousingthemousemodelofgranulocyticanaplasmosis.Specifically,wewilldetermine ifimmunizingagainsttheadhesins?bindingdomainsprotectsagainstsyringe-andtick-transmittedAp challenge.DoingsowillyieldacomprehensiveunderstandingofApinfectionandestablishasound rationalefordevelopingapproachesforpreventingHGA.